A toxin-deformation dependent inhibition mechanism in the T7SS toxin-antitoxin system of Gram-positive bacteria

Toxin EsaD secreted by some S. aureus strains through the type VII secretion system (T7SS) specifically kills those strains lacking the antitoxin EsaG. Here we report the structures of EsaG, the nuclease domain of EsaD and their complex, which together reveal an inhibition mechanism that relies on significant conformational change of the toxin. To inhibit EsaD, EsaG breaks the nuclease domain of EsaD protein into two independent fragments that, in turn, sandwich EsaG. The originally well-folded ββα-metal finger connecting the two fragments is stretched to become a disordered loop, leading to disruption of the catalytic site of EsaD and loss of nuclease activity. This mechanism is distinct from that of the other Type II toxin-antitoxin systems, which utilize an intrinsically disordered region on the antitoxins to cover the active site of the toxins. This study paves the way for developing therapeutic approaches targeting this antagonism.

lines 80-81: "However, secondary structure prediction indicated neither intrinsic disordered region nor DNA binding domain in EsaG" While the crystal structures confirm these statements, it should be noted that this cannot be derived from prediction. For protein segments that fold upon binding, both secondary structure predictors based on sequence as well as Alphafold2 tends to predict the folded state. Also disorder predictors do not recognize folding-upon)binding IDP domains of antitoxins as unfolded.
lines 94-95: "we launched a systematic effort to solve the structures ..." > "we determined the structures ...". Solvinf three structures is not a systematic effort.
line 95: "of the nuclease domain" -> "of the nuclease domain of EsaD" line 111: "The conformation of EsaDc is out of expectation in the EsaDc-EsaG complex structure". This is incorrect English. Furthermore, one should not talk about a "complex structure" (which means that the structure is very complicated" but about "the structure of the complex". Please correct this throughout the text. line 112: "asymmetric unit cell" -> "asymmetric unit" (an "asymmetric unit cell does not exist", you talk either about the asymmetric unit or about the unit cell, which are two distinct things that should not be mixed) line 119: "the central 6-stranded beta sheet of EsaDc is split in two separate subunits". It is NOT split in SUBUNITS (that is a wrong term here). You should just write that "it is split". line 121: "from the right side of β4'". There is no such thing as a right or a left side. Nor a top and bottom or back and front. This depends on the orientation of your molecule, and there is no universal reference frame. Therefore, this type of description is meaningless. line 136: "All the secondary structures" -> "All the secondary structure elements" line 163: you either "performed A His-tag pull down assay" or you "performed His-tag pull down assayS" Same line 170: "We also performed A cross-linking assay" or "We also performed cross-linking assayS" depending on whether there was one or more than one different cross-linking assay Line 168: I do not understand how a pull-down assay can tell you anything about the stoichiometry and conformational changes that occur upon interaction between EsaD and EsaG. The cross-linking mass spec experiments are more revealing in this respect, but to be really convincing, SAXS analysis of free proteins and the complex need to be performed as well.
Lines 184-186: "Another discovery in the pull-down assay is the association between the NT and the CT (Fig 2d), we thus deduced that these two fragments may reassemble into an intact structure of EsaDc in the absence of EsaG" This is not really surprising as it is the case for many proteins to produce a correctly folded structure out of two fragments. For example, a number of applications using GFP in cell biology are based on this property. Spending a whole paragraph on this is therefore not warranted.
lines 205-206: What do you mean with "is well preserved in the crystal lattice"? Do you mean the asymmetric unit contains a monomer and the dimer is formed according to crystal symmetry? If so, rephrase correctly. If you mean something else, also rephrase so the reader knows what you mean. In any case, confirmation of the dimer arrangement via SAXS is required. line 220: "Each of the EsaG structure in the dimer ..." This reads like in the dimer, the two EsaG monomers adopt different conformations. I think you mean "Each of the EsaG monomers in the dimer ..." lines 223-224: "However, severe steric clash occurs between Subunit1 and the unoverlapped EsaG in the homodimer ( Fig S9)" I do not understand this sentence. Wat is "Subunit1"? It reads like in your crystal structure the two EsaG monomers that make up the EsaG dimers clash with each other, which is a contradiction.
Reviewer #2 (Remarks to the Author): This manuscript presents a structure function study of a toxin antitoxin complex from the staphylococcal T7SSb. The authors provide an in-depth understanding of the molecular mechanism, by which the antitoxin EsaG inhibits the nuclease toxin EsaD. It is a very nice study which makes an important contribution to the T7SS field. Comments: -the authors should briefly explain why they used a truncated version of EsaD for their crystallization studies and subsequently for all functional assays. Does the proposed inhibition mechanism also work with full-length EsaD?
-In the prey cell EsaG is supposed to neutralize EsaD. How fast are EsaG-EsaDc complexes formed and what is the association rate? -in the cytoplasm the EsaD-EsaG complex is bound by EsaE. Could this have an effect on complex formation/inhibition? -The proposed heterotetramer formation is interesting but does not contribute to the inhibition mechanism itself. Could the authors speculate on its potential biological relevance in the discussion? -Pull down assay: it is unclear to me what was loaded onto the NiNTA column: purified proteins or cell lysates containing the overexpressed proteins. The figure legend for 2d) could use a description.
-the Ramachandran Plot statistics should also be provided in table 1.
-the labeling is a bit inconsistent and could also be clearer. Subunit 1/2 would be better labeled as EsaDc-Subunit1/2 or perhaps better EsaDc subdomain 1/2. Pull-down assay: Perhaps use His-EsaDc-Subdomain 1/2 instead of His-Sumo-Nt/Ct as label.
-the sentence in lines 79-82 should be reformulated. This sounds like as if secondary structure predictions can be used to predict DNA binding sites. Reviewer #3 (Remarks to the Author): In the presented paper, the authors report the structure of EsaD separate, and in complex with EsaG. It is shown that significant structural rearrangements of the EsaD's c-terminal occur upon binding to EsaG. Although the results on the interactions between EsaD and EsaG are interesting, I don't think they are significant enough for Nature communications. Therefore, the paper would be a better fit in a more specialized journal. Finally, I have some comments below that should be addressed before publication.
1. The authors only crystallize the c-terminal of EsaD with and without EsaG. They should motivate how we can be sure that these structures are representative for the full EsaD-EsaG complex.
2. The statements about the polymerization states of EsaG and EsaD through the paper can be a bit confusing. In the introduction the authors say that in the free state EsaD is a monomer and EsaG is a dimer. But in the bound state, EsaD is a dimer and EsaG is a monomer, therefore the complex should be a heterotrimer right? This is not consistent with the results paper of the section. For example in "Dimer to monomer conversion of of EsaG during EsaDc-EsaG interaction" the authors say that the EsaDc-EsaG is a heterotetramer according to gel-filtration experiments and that two EsaDc-EsaG complexes might interact. I believe the authors suggest an EsaG-EsaD-EsaD-EsaG arrangement, but then it should have been made clear in the introduction.
Comments on MD simulation: 3. The error bars for the energy profiles from Umbrella sampling look really large ( Figure 4C). I suspect this is because the simulation length for each window are really short, only 5 ns. They should probably be around 10-15 ns to improve the errors.
4. The authors state that boostrap analysis was used to calculate the errors, but they need to give more details. There are several approaches to do this for umbrella sampling, and some can underestimate the errors. See for example this publication https://pubs.acs.org/doi/pdf/10.1021/ct100494z. 5. The authors say that equilibration simulations were done at the temperature of 277 K and don't say anything about the temperature in the umbrella sampling simulations. What was the temperature in these simulations? If the temperature in the umbrella sampling simulations was also 277 K the authors should motivate this choice, considering the structural changes are expected to occur at room temperature in vivo.
6. The authors should report the force constant that was used for window restraints in the umbrella sampling simulations.
7. Finally, the CHARMM36 protein force field have been upgraded to CHARMM36m in 2017 and should be used in future simulations.
We thank all the reviewers for their critical comments on our work. We tried our best to address all their concerns and revised the manuscript accordingly. Please find our point-topoint response to each of the reviewers' comments below.

Reviewer #1 (Remarks to the Author):
While this is an interesting finding, little supporting information is presented that would strengthen the conclusions and reject alternative hypotheses. Furthermore, the paper is plagued by poor and inaccurate descriptions, that make it difficult to understand what exactly happens and whether the data are interpreted correctly. Although the last (schematic) figure is revealing in this respect (they are not numbered in the document), the text itself is often confusing, in part because of poor English and the use of incorrect terminology (for examples see below). In absence of the crystal structures (which were not made available for refereeing) it is always difficult to establish if the conclusions drawn from a structure are likely correct or not (the validation reports say something about the quality of the structure itself, but give no clue if it is correctly interpreted in terms of biology). Missing is validation of the structures using SAXS given that true interfaces of the complexes need to be distinguished from lattice contacts.
Response ： We greatly appreciate the reviewer's constructive suggestions and critical comments on our manuscript, and also realize the inefficiency of supporting information to reach the conclusions. We have included the following work in the revised manuscript: 1) We performed different assays to investigate the oligomer state of EsaG. Even though the crystal structure of EsaG and gel-filtration analysis imply a dimer state, other experiments including chemical cross-linking and density gradient centrifugation support a monomer form. Actually, we also solved the structure of another EsaG-like protein, YezG, which is an antitoxin to inhibit the nuclease activity of the toxin YeeF in the Gram-positive bacterium Bacillus subtilis. YezG shares about 40% sequence identity with EsaG. It is also eluted from gel-filtration column at a dimer-size position. However, not dermic interface was found in the YezG structure ( Fig. 1 in next page). The different packing patterns of EsaG, YezG and BH3703 further indicate that EsaG should be a monomer in solution. We also tried to follow the review's suggestion to perform SAXS at Shanghai Synchrotron Radiation Facility, but our appointment was postponed indefinitely because of the outbreak of COVID-19 in Shanghai. We are really sorry for that, and hope our alternative experiments could satisfy the reviewer.
2) For the EsaDc-EsaG tetramer, we performed native mass spectrum, cross-linking and 19 F-NMR studies. The results confirm the existence of tetramer state of EsaDc-EsaG complex is solution. Additionally, the cross-linking assay and 19 F-NMR studies revealed that it is the CFs that involve in the tetramer formation, but not the NFs.
We have revised our model accordingly.

3) We performed functional studies on the finger loop of EsaG and MD studies on the process of EsaDc-EsaG interaction, which together revealed that the finger loop is
the trigger of the conformational changes on EsaDc induced by EsaG.

4) We performed His-tag pull-down and in vitro enzymatic assays on another toxin,
YeeF, to show that YeeF could also be split into two separated fragments, which can interact with YezG independently and reassemble into a functional protein in the absence of YezG. These results support that the inhibition mechanism discovered in this paper might be widely used by Gram-positive bacteria.

5)
We have gone through the manuscript carefully to correct the inaccurate descriptions, and reorganized the results to better support our conclusions.
The conformational changes that are reported, are certainly complex and it is unlikely that these could be predicted. However, there is at least one other TA system where (other but) equally large The YezG structure is not included in this paper.
changes in the toxin are observed upon binding the antitoxin, and also in that case an alternative mode of dimer formation of the toxin is observed: E. coli rnlAB (Garcia-Rodriguez et al, 2021, NAR 49(12):7164-7178). This work is not cited nor discussed as it should be.
Response：We thank the reviewer for pointing this out. We have cited this paper in the revised manuscript as "A recent study on RnlA-RnlB, a type II toxin

Response：We have revised the manuscript as suggested.
lines 80-81: "However, secondary structure prediction indicated neither intrinsic disordered region nor DNA binding domain in EsaG" While the crystal structures confirm these statements, it should be noted that this cannot be derived from prediction. For protein segments that fold upon binding, both secondary structure predictors based on sequence as well as Alphafold2 tends to predict the folded state. Also disorder predictors do not recognize folding-upon)binding IDP domains of antitoxins as unfolded.
Response：We have removed this sentence from the revised manuscript.
lines 94-95: "we launched a systematic effort to solve the structures ..." > "we determined the structures ...". Solvinf three structures is not a systematic effort.

Response：We have revised this sentence as "we next solved the crystal structures of EsaD and the EsaD-EsaG complex".
line 95: "of the nuclease domain" -> "of the nuclease domain of EsaD" Response: We have revised the manuscript as suggested.
line 111: "The conformation of EsaDc is out of expectation in the EsaDc-EsaG complex structure". This is incorrect English. Furthermore, one should not talk about a "complex structure" (which means that the structure is very complicated" but about "the structure of the complex".
Please correct this throughout the text.

Response: We have revised the manuscript as suggested.
line 112: "asymmetric unit cell" -> "asymmetric unit" (an "asymmetric unit cell does not exist", you talk either about the asymmetric unit or about the unit cell, which are two distinct things that should not be mixed)

Response: We have revised the manuscript as suggested.
line 119: "the central 6-stranded beta sheet of EsaDc is split in two separate subunits". It is NOT split in SUBUNITS (that is a wrong term here). You should just write that "it is split".

Response: We thank the reviewer for pointing this out. We used NF (N-terminal fragment of EsaDc) and CF (C-terminal fragment of EsaDc) in the revised manuscript to replace
Subunit1 and Subunit2, respectively. line 121: "from the right side of β4'". There is no such thing as a right or a left side. Nor a top and bottom or back and front. This depends on the orientation of your molecule, and there is no universal reference frame. Therefore, this type of description is meaningless.

Response: We have revised the manuscript as suggested.
line 136: "All the secondary structures" -> "All the secondary structure elements" Response: We have revised the manuscript as suggested.
line 163: you either "performed A His-tag pull down assay" or you "performed His-tag pull down assayS" Response: We have revised the manuscript as "performed a His-tag pull down assay".
Same line 170: "We also performed A cross-linking assay" or "We also performed cross-linking assayS" depending on whether there was one or more than one different cross-linking assay

Response: We have revised the manuscript as suggested.
Line 168: I do not understand how a pull-down assay can tell you anything about the stoichiometry and conformational changes that occur upon interaction between EsaD and EsaG.
The cross-linking mass spec experiments are more revealing in this respect, but to be really convincing, SAXS analysis of free proteins and the complex need to be performed as well.
Response: We are sorry for the misleading language in this part. The cross-linking assay and pull-down assay were designed to validate that EsaDc interact with EsaG through two separated fragments, as observed in the EsaDc-EsaG heterodimer. We have reorganized sentences in this part to make it clear.
Lines 184-186: "Another discovery in the pull-down assay is the association between the NT and the CT (Fig 2d), we thus deduced that these two fragments may reassemble into an intact structure of EsaDc in the absence of EsaG" This is not really surprising as it is the case for many proteins to produce a correctly folded structure out of two fragments. For example, a number of applications using GFP in cell biology are based on this property. Spending a whole paragraph on this is therefore not warranted.
Response: We agree with the reviewer that many proteins have been found to produce a correctly folded structure out of two fragments. Following the reviewer's suggestion, we have shortened this part by removing the result of gel-shift experiment.
But we believe this is an important part to the inhibitory mechanism proposed in our paper. During secretion process, EsaG is removed from the complex. However, we don't know whether the two separated fragments reassemble into a functional protein spontaneously or with the help of other proteins. The results of our experiments, including pull-down assay, NMR experiment and enzymatic activity assay, lend strong support to the spontaneous assemble of EsaDc fragments.
lines 205-206: What do you mean with "is well preserved in the crystal lattice"? Do you mean the asymmetric unit contains a monomer and the dimer is formed according to crystal symmetry? If so, rephrase correctly. If you mean something else, also rephrase so the reader knows what you mean. In any case, confirmation of the dimer arrangement via SAXS is required.
Response: 1) We removed this sentence from the revised manuscript. 2) As mentioned above, we tried to follow the review's suggestion to perform SAXS at Shanghai Synchrotron Radiation Facility, but our appointment was postponed indefinitely because of the outbreak of COVID-19. It is still shut down now. We therefore performed chemical cross-linking and density gradient centrifugation experiments, which supported a monomer form of EsaG in solution. We thank the reviewer sincerely for pointing this out, which help us correct the error in our paper. I do not understand this sentence. Wat is "Subunit1"? It reads like in your crystal structure the two EsaG monomers that make up the EsaG dimers clash with each other, which is a contradiction.
Response: "Subuint1" refers to the N-terminal fragment of EsaDc. Since we confirmed the monomer state of EsaG, the sentence mentioned above has been removed from the new manuscript.

Reviewer #2 (Remarks to the Author):
This manuscript presents a structure function study of a toxin antitoxin complex from the staphylococcal T7SSb. The authors provide an in-depth understanding of the molecular mechanism, by which the antitoxin EsaG inhibits the nuclease toxin EsaD. It is a very nice study which makes an important contribution to the T7SS field.
Response: We appreciate the positive comments of this reviewer on our manuscript. Our responses to the reviewer's advice on the minor points/questions are listed below. Comments: -the authors should briefly explain why they used a truncated version of EsaD for their crystallization studies and subsequently for all functional assays. Does the proposed inhibition mechanism also work with full-length EsaD?

Response: We thank the reviewer for pointing this out. EsaD contains two domains: the Nterminal domain of EsaD interacts with a chaperon protein, EsaE, and they together play critical roles in the secretion of EsaD through T7SS; the C-terminal domain of EsaD encodes a typical ββα-metal nuclease, which interacts with the antitoxin EsaG. It has been
shown that the interaction of EsaG with the nuclease domain oF EsaD is sufficient to neutralize the toxicity of (Nat. Microbiol., 2016, 2, 16183). We also clarified this in the revised manuscript.
-In the prey cell EsaG is supposed to neutralize EsaD. How fast are EsaG-EsaDc complexes formed and what is the association rate?

Response: We tested the binding affinity between EsaD and EsaG by BLI. Ths K d is about
0.46 µM. In fact, EsaD is highly toxic to the host cells. The wild-type EsaD gene is even unable to be cloned in E. Coli., so the protein has to be neutralized immediately upon synthesis.
-in the cytoplasm the EsaD-EsaG complex is bound by EsaE. Could this have an effect on complex formation/inhibition? Response: It has been reported that EsaE does not interact with EsaG or the nuclease domain of EsaD (Nat. Microbiol., 2016, 2, 16183).
-The proposed heterotetramer formation is interesting but does not contribute to the inhibition mechanism itself. Could the authors speculate on its potential biological relevance in the discussion?
Response: We tried to figure out the biological functions of the heterotetramer formation by mutagenesis, but failed to obtain any mutant that disrupt the dimeric interface of EsaD while at the same time keep the protein stable. We speculate that the heterotetrameric arrangement may help keep EsaDc in the inhibited state by stabilizing the exposed hydrophobic residues on CF. We have added this in the Discussion as suggested.
-Pull down assay: it is unclear to me what was loaded onto the NiNTA column: purified proteins or cell lysates containing the overexpressed proteins. The figure legend for 2d) could use a description.
Response: Thank you pointing this out. Purified proteins were used to perform the pulldown assays. We have added this information in the corresponding figure legend.
-the Ramachandran Plot statistics should also be provided in table 1. Table S1 as suggested.

Response: The Ramachandran Plot statistics have been provided in
-the labeling is a bit inconsistent and could also be clearer. Subunit 1/2 would be better labeled as EsaDc-Subunit1/2 or perhaps better EsaDc subdomain 1/2. Pull-down assay: Perhaps use His-EsaDc-Subdomain 1/2 instead of His-Sumo-Nt/Ct as label.

Response:
We are sorry about this problem. In the revised manuscript, we replaced Subunit1/2 with NF/CF, because another reviewer disagree that EsaD is split into "subunits". As suggested, we labeled samples in the pull-down assay as His-EsaDc-NF/CF. Response: We have revised these errors.

Reviewer #3 (Remarks to the Author):
In the presented paper, the authors report the structure of EsaD separate, and in complex with EsaG. It is shown that significant structural rearrangements of the EsaD's c-terminal occur upon binding to EsaG. Although the results on the interactions between EsaD and EsaG are interesting, I don't think they are significant enough for Nature communications. Therefore, the paper would be a better fit in a more specialized journal. Finally, I have some comments below that should be addressed before publication.
Response：We are grateful to the reviewer for his/her advices on our manuscript. However, we want to stress that our finding is not just interesting, but also represents a major conceptual advance on the toxin-antitoxin systems. Secondly, the conformational changes during the EsaDc-EsaG interaction is complicate, and is "unlikely that these could be predicted", as pointed out by reviewer 1. The structures determined in our paper provide new examples to study the plasticity and dynamic of proteins, which is not only attractive to researchers studying the structure-function of macromolecules, but also presents a challenge to theoretical experts who try to predict protein-protein interactions. In the revised manuscript, we performed ratchet&pawl potential and REST2 to get insights into the mechanism that is difficult to understand solely from experiments. But our work only revealed what happens at the very early stage of EsaD-EsaG association, how the β-sheet in the core of EsaDc is split remains elusive. We are still looking for top professionals on MD to analyze the process.
Thirdly, multidrug resistance among Gram-positive bacteria, especially methicillinresistant S. aureus (MRSA), has been a major healthcare concern worldwide. Toxinantitoxin modules usually play critical roles in persister formation. The inhibitory mechanism revealed in this study therefore sheds light on development of novel antibiotics that release EsaD from the complex to kill the bacteria. Furthermore, EsaD is the first antibacterial toxin identified to secrete through the T7SS of S. aureus. The structure of EsaDc-EsaG complex will help to design EsaD mutants that are insensitive to EsaG, which may also be used to treat or prevent S. aureus infection. As pointed out by reviewer 2, this would make "an important contribution to the T7SS field".
Together, we believe that our finding is scientifically significant and will be of broad interest in readers of Nature Communications. The newly added data and revisions have greatly strengthened our manuscript. We hope that the reviewer could reconsider our manuscript.
1. The authors only crystallize the c-terminal of EsaD with and without EsaG. They should motivate how we can be sure that these structures are representative for the full EsaD-EsaG complex.
Response: We understand the reviewer's concern and thank the reviewer for pointing this out. EsaD contains two domains: the N-terminal domain of EsaD interacts with a chaperon protein, EsaE, and they together play critical roles in the secretion of EsaD through T7SS; the C-terminal domain of EsaD encodes a typical ββα-metal nuclease, which interacts with the antitoxin EsaG. It has been shown that the interaction of EsaG with the nuclease domain oF EsaD is sufficient to neutralize the toxicity of (Nat. Microbiol., 2016, 2, 16183).
We also clarified this in the revised manuscript.
2. The statements about the polymerization states of EsaG and EsaD through the paper can be a bit confusing. In the introduction the authors say that in the free state EsaD is a monomer and EsaG is a dimer. But in the bound state, EsaD is a dimer and EsaG is a monomer, therefore the complex should be a heterotrimer right? This is not consistent with the results paper of the section.
For example in "Dimer to monomer conversion of of EsaG during EsaDc-EsaG interaction" the authors say that the EsaDc-EsaG is a heterotetramer according to gel-filtration experiments and that two EsaDc-EsaG complexes might interact. I believe the authors suggest an EsaG-EsaD-EsaD-EsaG arrangement, but then it should have been made clear in the introduction.